Project summary: Infectious diseases that disproportionately impact impoverished regions in the tropics have devastating impacts on human health and economic development, with billions of people at risk. These diseases include HIV/AIDS, malaria, and tuberculosis, but also the neglected tropical diseases (eg, schistosomiasis, filariasis, and other helminth, microbial, and protozoan infections). The agents that cause these diseases are often co-endemic. Concomitant infections can alter host responses, disease prognosis, transmission dynamics, and treatment outcomes, as well as posing difficulties for accurate interpretation of data focused on single infections. A single, simple diagnostic test that could specifically and concurrently detect those infections present in each individual with high sensitivity would be a breakthrough in terms of cost, time, and accurate diagnosis. Unfortunately, current diagnosis and monitoring of disease transmission and efficacy of treatment depend largely on methods that are often inaccurate, labor-intensive, or unreliable. These limitations acquire added significance in mass drug administration programs, where measures of effectiveness require accurate monitoring of infection (and coinfection) status, treatment success, disease transmission rates, and emergence of drug resistance. Molecular detection of pathogen nucleic acids in host fluids or tissues offers reliability, sensitivity, and specificity, but current methods require infrastructural support, highly- trained staff, and expensive, delicate equipment. In this project, we will build upon our substantial advances in development of microfluidic, point-of-care/contact (POC) devices that enable on-site, inexpensive molecular diagnosis of infection and monitoring of disease transmission by minimally-trained personnel. Specifically, we will: Aim 1) develop, optimize, and validate a new 2-stage amplification pathogen detection protocol (RAMP) using benchtop assays; and Aim 2) transfer and adapt the benchtop RAMP assays developed in Aim 1 to a new microfluidic, multiplexing chip, and verify that the chip allows for simultaneous POC detection of multiple pathogen nucleic acids. Accomplishment of these aims will serve as proof of concept and set the stage for more extensive refinement and testing in future work using animal models of infection and coinfection, clinical samples, and field studies. The approach will readily translate into groundbreaking new technology for field-ready, POC molecular diagnosis and monitoring of tropical diseases and coinfections.